Citrobacter freundii strain WA1 was stressed by incubation in seawater microcosms for eleven years. After two years of starvation, no culturable strain was observed. Incubation of samples in nutrient-rich broth medium not supplemented with growth factors, however, allowed resuscitation of VBNC cells so that subsequent plating yielded observable colonies for significantly extended periods of time. Recovery of VBNC Citrobacter freundii was obtained by incubation in nutrient broth even after eleven years of starvation. To see whether the samples contained the same strain of Citrobacter freundii inoculated 11 years ago. The complete 16S rRNA gene was PCR amplified and sequenced from initial, stressed and revived strains of Citrobacter freundii strain WA1.The 16S rRNA gene sequences from eleven-year stressed strains were homologous with a high degree of similarity to the GenBank reference strain and were identical to each other.

Salmonella enterica serovar Typhimurium was stressed by incubation in seawater and soil microcosms for 20 years. Recovery of viable but nonculturable Salmonella was obtained by incubation in nutrient broth and buffered peptone water medium for 2 months, and also in a mouse model when administered orally but not by intra-peritoneal injection. The 16S rDNA gene sequences from 20-year stressed strains were homologous, with a high degree of similarity, and all were identical to the respective initial strain.

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Salmonella enterica serovar typhimurium was stressed by incubation in seawater microcosms for seventeen years. The microcosms were prepared in such a way as to allow progressive evaporation of the water. Despite being introduced into the sterile seawater at very high concentrations, the Salmonella rapidly declined to levels undetectable by plate counts on nutrient agar. After two years of starvation, about half of the seawater volume had evaporated from each microcosm, and salt crystals appeared. Inoculation of the salty suspension did not result in any culturable strains in selective and non-selective media. However, incubation of samples in nutrient-rich broth, without supplemental growth factors, allowed resuscitation of stressed cells, yielding colonies that remained viable for extended periods of time. After three years the total volume of water had evaporated from each microcosm, and only salt crystals remained. These microcosms were then incubated at room temperature for seventeen years. Resuscitation of VBNC Salmonella from the salt crystals was conducted in vitro and in vivo; recovery occurring after incubation in both nutrient broth and in mice. Recovery occurred in the Salmonella